Molecular identification of Trypanosoma theileri (Laveran, 1902) in cattle from two slaughterhouses in Ecuador and its relation with other haemotropic agents.

Trypanosoma theileri is a worldwide distributed haemoparasite that has been reported throughout the American continent in various species, including bovines, buffaloes and bats. In bovines, high incidence of T. theileri can be harmful when associated with other infections or under stress situations. There is little information on this hemoflagellate in Ecuador, which prompted the study and molecular identification of the trypanosomes collected in two slaughtering centers. Between February and April 2021, a total of 218 samples of bovine blood were collected in abattoirs located in the Andean region of Quito (n = 83) and in the coastal region, in Santo Domingo (n = 135). Quito public Slaughterhouse is the biggest in Ecuador, and for that, they receive animals from all country; on the other hand, Santo Domingo's Slaughterhouse is a small one where mainly females from the region are sacrificed and some males. The samples were evaluated using two molecular tests, the PCR cathepsin L-like (CatL) specific for T. theileri and for the positive samples, a Nested PCR that targets the ITS of the 18S gene. The corresponding PCR products were sequenced, analyzed by BLAST/NCBI and the sequences were used to build a concatenated phylogenetic tree, using the MEGA XI software. Overall, 34 out of the 218 samples, (15.6%) were positive to T. theileri by PCR CatL, resulting from 20/83 (24.1%) positives from the Quito abattoir and 14/135 (10.4%) from the Santo Domingo slaughterhouse. These prevalence rates were found to be significantly different (p = 0.006). According to the phylogenetic tree based on the CatL and ITS concatenated sequences (n = 13), the two novel Equatorial T. theileri isolates, ThI (n = 7) and ThII (n = 6) are closely related and associated to the IC, IB and IIB genotypes, present in Brazil, Venezuela and Colombia. Thirty-one out of the thirty-four T. theileri-positive bovines were co-infected with other haemotropic pathogens, Anaplasma marginale Babesia spp and T. vivax. This coinfection could be responsible for additional pathologies and harmful effects on the affected cattle. This study presents the molecular identification and genotypification of T. theileri isolated from cattle in Ecuador through the analysis of CAtL and ITS sequences, and the high frequency of coinfection of this hemoflagellate with other blood haemotropic organisms.

First Molecular Identification of Trypanosomes and Absence of Babesia sp. DNA in Faeces of Non-Human Primates in the Ecuadorian Amazon.

Trypanosomes are a group of pathogens distributed in the continents of Africa, America, Asia and Europe, and they affect all vertebrates including the neotropical primate group. Information about the trypanosome's diversity, phylogeny, ecology and pathology in non-human primates (NHPs) from the neotropical region is scarce. The objective of the study was to identify Trypanosoma and Babesia molecularly in NHPs under the phylogenetic species concept. We extracted DNA from a total of 76 faecal samples collected between 2019 and 2021, from a total of 11 non-human primate species of which 46 are from captive NHPs and 30 are free-living NHPs in the Western Amazon region of Ecuador. We did not detect DNA of Babesia sp. by polymerase chain reaction test in any of the faecal samples. However, the nested-PCR-based method revealed Trypanosoma parasites by ITS gene amplification in two faecal samples; one for the species Leontocebus lagonotus (from the captive population) and a second one for Cebus albifrons (from the free-ranging population). Maximum parsimony and likelihood methods with the Kimura2+G+I model inferred the evolutionary history of the two records, which showed an evolutionary relationship with the genus Trypanosoma. Two sequences are monophyletic with Trypanosoma. However, the number of sequences available in GenBank for their species identification is limited. The two samples present different molecular identifications and evolutionary origins in the tree topology. We are most likely referring to two different species, and two different localities of infection. We suggest that health management protocols should be implemented to prevent the transmission of blood-borne pathogens such as Trypanosoma sp. among captive populations. In addition, these protocols also protect the personnel of wildlife rehabilitation centers working in close proximity to NHPs and vice versa.

First report and molecular identification of Trypanosoma (Duttonella) vivax outbreak in cattle population from Ecuador.

The bovine trypanosomosis is responsible for economic losses from tropical and subtropical areas of Africa and Latin America. This disease is characterized by fever, anaemia, loss of production and even death. Few studies have been carried out in Ecuador regarding Trypanosoma spp. presence but the species has not been determined in cattle and those have only determined the presence of genus, but not the species. The aim of this study was to identify and characterize the trypanosome species involved in the suspected bovine trypanosomosis outbreak reported in Convento Village in Manabí Province located in the coastal region of Ecuador. Twenty cattle from three farms were sampled. Three samples were positive for T. vivax, using an end-point polymerase chain reaction (PCR) to amplify a fragment of the cathepsin L-like cysteine protease (CatL-like) gene. A phylogenetic tree analysis of these three Ecuadorian isolates showed a close relationship with isolates from South America (Colombia, Brazil and Venezuela) and West Africa (Nigeria). This is the first report of T. vivax in Ecuadorian cattle.

PCR-diagnosis of Anaplasma marginale in cattle populations of Ecuador and its molecular identification through sequencing of ribosomal 16S fragments.

BACKGROUND: Bovine anaplasmosis is an endemic disease in tropical and subtropical areas. It is caused by a bacterium named Anaplasma marginale, and represents an economic problem for cattle farmers due to the losses it generates, such as: mortalities, reduced production, quarantine measures, treatments and control of vectors. The method most often used to diagnose this haemotrophic bacterium is direct examination on blood smear, which sensitivity and specificity are limited compared to other methods such as PCR. The present study aimed at investigating the presence of A. marginale in dairy cattle of Luz de América commune, province of Santo Domingo de los Tsachilas. Two PCRs were used to amplify specific regions of the Rickettsia for its molecular identification. RESULTS: At first, 151 blood samples were tested: msp5 specific gene of A. marginale was identified in 130 samples, meaning 86.1% of them were infected by the rickettsia. Two positive samples were further randomly selected to confirm the presence of A. marginale through amplification, cloning and sequencing of the conserved region of gene 16S rRNA. The analysis of sequences obtained through cloning revealed a 100% identity between both samples and those registered in GenBank for A. marginale. CONCLUSION: This is the first report and molecular identification of A. marginale in the bovine population of Ecuador and its prevalence was high at the level of farms and animals. These results demonstrate the importance of proceeding to evaluate and characterize bovine Anaplasmosis in Ecuador in order to establish control measures and reduce their impact.